Project Summary The goal of this research project is to understand the molecular aspects of Nfatc1 in developmental coronary angiogenesis (DCA). We attempt to achieve the goal by using a synergistic approach of mouse genetics, developmental and molecular biology, and systems biology. DCA by the ventricular endocardial cells (VECs) gives rise to coronary arteries; therefore, successful completion of the proposed research will have significant translational implications by revealing the factors underlying DCA that can be relevant to the etiology of congenital coronary artery defects, one of major causes of sudden death. Better understanding of DCA will also provide the developmental basis that is critically needed for cell-based therapies for coronary heart disease, the leading cause of heart failure and death. DCA is a cardiac-specific process different from the vascular angiogenesis in other organs of body. Factors underlying this process are understudied and remain largely unknown due to the lack of proper animal models and experimental approaches. This project aims to fill the gap by studying newly generated mouse models of defective DCA and coronary arteries, focusing on an endocardial specific transcription factor, nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1) in the regulation of a VECs specific gene regulatory network (GRN) of DCA. Preliminary studies revealed that Nfatc1 is required for maintaining the expression of genes for the hematopoietic stem cells and multi-potent cardiovascular progenitor cells and limiting the expression of genes involved in endothelial differentiation and/or specification, and knockout of Nfatc1 leads to abnormal DCA. Based on these results, we hypothesize that Nfatc1 functions as a `molecular checkpoint' for timing DCA through its transcriptional regulation of a tissue specific GRN for the differentiation of progenitor VECs into coronary arterial endothelial cells. Three Aims are designed to test this hypothesis. Aim 1 will characterize the angiogenic VEC in the developing heart by their changes in the expression of angiogenic genes and cell shape, and functional angiogenesis assays. Aim 2 is to ascertain Nfatc1 functions in DCA and determine whether it interacts with the Vegf-Notch pathway that is critical for angiogenesis using mouse genetics, and in vitro functional assays. Aim 3 will identify the Nfatc1-dependent GRN and hub genes VECs using RNA-seq/ChIP-seq and bioinformatics analysis, and confirm their roles in DCA in vivo by expression and in vitro by angiogenesis assays.